Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora.
A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I-P-X-P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5kb contained the complete sequence of the Cs-mnp1 gene, including 162bp and 770bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT-AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9kb was amplified using inverse PCR. A putative TATAA element was identified 5' of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.
Lobos
S
S
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile.
Larrondo
L
L
Salas
L
L
Karahanian
E
E
Vicuña
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D005800:Genes, Fungal / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008031:Lignin / Q000378:metabolism*; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000302:isolation & purification; D020072:Polyporales / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D008031:Lignin; D010544:Peroxidases; C051129:manganese peroxidase